Culture Media Preparation

Culture media, also known as growth media, are specific mixtures of nutrients and other substances that support the growth of microorganisms such as bacteria and fungi (yeasts and molds). Culture media are used for quality control tests of nonsterile raw materials and finished products as well as for microbial contamination (sterility) tests in applications such as hygiene monitoring, sterilization process validation and determination of the effectiveness of preservatives and antimicrobial agents.

Microbial enumeration tests for nonsterile products are performed according to the harmonized test procedures of the European, US and Japanese pharmacopoeias. These tests, formerly known as Microbial Limit Tests (MLT), determine the bioburden of the product sample. This is achieved by counting the number of colony forming units that have grown on the culture media for a known sample size (CFU/g). Acceptance criteria are based on the Total Aerobic Microbial Count (TAMC), the Total Yeast and Mold Count (TYMC). Furthermore, specific tests are undertaken to confirm the presence or absence of certain microorganisms that may prove to be objectionable.

The culture media ingredients vary according to the test being performed and the microorganism of interest. Most commonly, culture media are based on a nutrient broth (liquid), often mixed with agar and prepared in petri dishes (they may be semi-solid or solid). For enriched or selective media, further specific components are added.

Culture media preparation must be carried out accurately to ensure microbiological growth is correctly promoted. The individual ingredients of the culture media (powders, gels and liquids) must be carefully weighed out according to the culture media formulation recipe. A precision balance with readability from 1 mg up to 10 mg is typically used for the main components. An analytical balance may be required for weighing out trace elements such as copper and zinc. If a smaller amount of culture media is required, care must be taken to correctly recalculate the ingredient quantities and a balance with a higher readability may be required in order to fulfil accuracy requirements.

Accurate culture media preparation is essential to ensure that microbiological growth is correctly supported. Watch the video to see how METTLER TOLEDO's XPR precision balances not only helps ensure your growth media are formulated precisely, but also simplifies the process to save you time and effort.

Preparation of culture media formulations, including liquid growth media and culture media based on nutrient agar, is a common procedure in any microbiology laboratory. The culture media formulation process involves many steps and must be carried out with care to avoid cross contamination and ultimately protect the health of consumers.

1. Select culture media recipe from database
2. Recalculate ingredient quantities according to the required culture media volume
3. Weigh main ingredients into the container
4. Weigh trace ingredients on a high accuracy balance and add to the container
5. Pour in deionized water up to around 80% of the required volume
6. Mix to dissolve the ingredients, gentle heating may be required
7. Check the pH using a pH meter and adjust if necessary
8. Top up the culture media to the required volume
9. Label the container
10. Sterilize in autoclave
11. Document results

Types of microbiological quality control tests

Depending on the type of product and its purpose, there are three main categories of microbial quality control tests requiring the use of growth media:

Guidelines for microbial examination of non-sterile products

The microbial limits test procedures are performed to determine whether a non-sterile product complies with given specifications for microbial quality. Such non-sterile products include cosmetic, healthcare and pharmaceutical products that may be ingested, applied topically or inhaled. The efficacy of such products may be affected or even eliminated by the presence of certain microorganisms and, additionally, some microorganisms can adversely affect the health of the consumer. The microbial enumeration tests are a range of tests manufacturers can use to help ensure the bioburden of their finished goods remains within safe limits.

While many countries have their own guidelines for establishing the safety of non-sterile products, there is harmonization of the important standards in the European, US and Japanese markets as is shown in the table below.

Guidelines for sterility testing of sterile products

Sterility testing confirms that products are free from the presence of viable microorganisms and is of vital importance for medical devices, pharmaceuticals, tissue materials and any other materials that claim to be sterile or free from viable microorganisms. As well as identifying potential contamination, sterility testing is also used to determine antimicrobial effectiveness and to validate sterilization procedures.

Complex culture media recipes coupled with a large number of ingredients can make culture media preparation a time and labor-intensive procedure. Getting it right is essential to ensure consumer safety. If you need support with culture media preparation or would like advice on topics such as how to ensure SOPs are followed, electrostatics, or documentation and traceability, our team of experts is here for you. Do not hesitate to contact us for help or advice.

Culture media and growth media often consist of more than five ingredients, sometimes as many as 30. Such complex formulation processes demand a great deal of care and attention, and even the fastest laboratory technician may need 1.5 h to prepare such a mixture.

Culture media calculations

The typical culture media recipe produces 1 liter of culture media solution. When a volume other than 1 liter is required, it is necessary to recalculate all the ingredient quantities accordingly and record all the new values. Any error in the calculations or in logging the new values can result in a culture medium that does not perform as required, potentially leading to severe consequences.

Weighing and recording weight results

When following a culture media recipe, or the recalculated values, the laboratory technician must be sure that the right amount of each ingredient is used, and indeed that all ingredients have actually been added. This can often mean ticking the ingredients off a list as they are weighed in. Care must be taken to avoid transcription errors when logging the actual weights or when transferring them to a computer. The use of two balances in the culture media preparation process adds to the complexity, reduces efficiency and increases the risk of errors.

Weighing influences

Culture media ingredients may be weighed out into a variety of different containers ranging from weighing boats up to five liter beakers. All containers, as well as the balance itself, may be subject to external influences, e.g. movement of air, electrostatic charges or the magnetic field from a stirrer, which can give rise to difficulties such as taring the balance, drifting and unstable readings, and longer settling times. Such challenges extend the time to make the culture media, affecting productivity.

The difficulties of weighing in a safety cabinet or fume hood

The effects of the movement of air within a safety cabinet or fume hood can be alleviated through the use of a draft shield to protect the balance and the container. While a draft shield is an excellent way to prevent weighing errors, there are some practical drawbacks:

1. Opening and closing the draft shield doors is time consuming
2. Limited access to the weighing pan reduces ergonomics
3. Bulky and/or heavy containers can be difficult to handle
4. The draft shield is an additional item that must be cleaned

Documentation

Not only must the culture media be fully labelled to avoid any mix-ups, all data related to the culture media must be logged and stored securely for future reference and traceability. Handling large quantities of data manually is time consuming and carries a high risk of error.

METTLER TOLEDO's XPR precision balances offer a high level of accuracy and address many of the challenges of culture media preparation, simplifying processes and reducing the workload for laboratory technicians. With readabilities from 0.1 mg to 100 mg and capacity up to 10 kg, XPR precision balances are perfect for a wide range of culture media preparation applications.

Recipes directly on the balance

Up to 50 culture media preparation recipes can be saved on the balance as weighing methods, enabling technicians to quickly access the recipe needed and start working. Step-by-step instructions are provided on the balance display so it's easy for technicians to keep track of where they are in the culture media preparation process.

Automatic calculations and results storage

At the beginning of the culture media recipe, the technician simply enters the required volume and the quantities of the ingredients are recalculated automatically. The balance prompts the technician to weigh in each ingredient, saves the actual weight result and tares automatically before the next ingredient is added. There is no need to record results manually and the results can be printed out automatically at the end.

No draft shield needed

All XPR precision balances are equipped with the SmartPan™ weighing pan. SmartPan provides extraordinary stability by minimizing the effects of air currents on the weighing cell making it possible to weigh in with 1 mg readability without a draft shield. SmartPan enables results to be delivered up to twice as fast as a standard weighing pan.

Easy cleaning

Clean equipment is essential to reduce the risk of cross-contamination. The SmartPan weighing pan retains any spillages on the pan base, enabling safe disposal. The pan and the base are easily removed for cleaning in a dishwasher. A protective cover on the body not only protects the balance by helping to prevent spills from entering the weighing cell but the smooth surface is easy to clean and prevents build-up of dirt. Chemically resistant covers are available for the balance display that can be simply lifted off after use and disposed of to avoid cross-contamination.

Have you ever wished that weighing-in and documentation processes for media formulation were less complicated? Is a challenge of reproducing optimized media formulation familiar to you? Read our guide and learn how tailored instruments, software and accessories can support R&D labs in achieving a high degree of weighing reliability, media consistency, overall cost-effectiveness and product quality for its formulation processes.

METTLER TOLEDO's XPR precision balances can be connected to our proprietary LabX laboratory software to create a full workflow solution that offers comprehensive data management capabilities. A centralized database provides all connected users with access to the same SOPs for culture media preparation, ensuring that each technician follows the same procedures. The software takes care of all process information and calculations automatically. Results are saved securely in the database and can be accessed at any time for further analysis and report generation. The software practically eliminates the need for manual recording of data – even detailed labels can be printed automatically.

The solution can be integrated with existing ERP/LIMS systems, enabling bi-directional transfer of recipes and results. Instruments, tasks and users can all be managed centrally; laboratory managers can monitor the live progress of tasks without needing to be present in the laboratory. For highly regulated environments, the software helps with compliance with FDA ALCOA+ data integrity requirements.

Whether you just need rapid and reliable pH measurements or must fulfil stringent regulations, METTLER TOLEDO can support you.

The top-of-the-range SevenExcellence line offers intuitive OneClick™ touchscreen operation, enabling saved methods to be started directly from a shortcut on the home screen. Intelligent sensor management ensures your pH electrodes are always in top condition for accurate results. The Seven Excellence line can be connected to LabX to ensure traceability and data integrity.

For pH measurements on flat surfaces like agar plates, specialized surface electrodes like the InLab Surface Pro-ISM are recommended.